Evidence of human milk oligosaccharides in maternal circulation already during pregnancy: a pilot study.

Human milk oligosaccharides (HMOs) are bioactive glycans linked with health benefits to both the breast-fed infant and lactating mother. We hypothesized that HMOs are present before lactation, already during pregnancy, and are influenced by maternal body composition. In a pilot study, we investigated individual and temporal variations in HMO composition and concentration in maternal serum at gestational weeks 10-14 ( visit 1), 20-24 ( visit 2), and 30-35 (visit 3) (V1, V2, and V3, respectively) and associations with maternal body composition. HMOs were quantified by HPLC and confirmed by enzymatic digest and mass spectrometry. Associations of maternal prepregnancy body mass index (BMI), subcutaneous adipose tissue (SAT) thickness, and adipokines with absolute and relative HMO concentrations were analyzed by Spearman correlation. We identified 16 HMOs and 2 oligosaccharides not common to human milk. HMO concentration and composition varied with gestational age and secretor status. HMO concentration increased with gestational age and changed from a predominantly sialylated profile at V1 to a more balanced fucosylated-to-sialylated ratio at V3, mostly due to a profound increase in 2'-fucosyllactose (2'-FL), reflecting secretor phenotype. In secretor-positive women, BMI was negatively correlated with 2'-FL at V2. SAT at V1 and V2 were strongly negatively correlated with 2'-FL concentrations. This pilot study shows that prenatal HMOs are present in maternal serum, suggesting roles for HMOs already during pregnancy. Our result that maternal body composition is associated with prenatal HMOs might indicate that maternal metabolism modulates HMO composition with unknown implications for maternal and fetal health already during pregnancy.

A hormone-dependent feedback-loop controls androgen receptor levels by limiting MID1, a novel translation enhancer and promoter of oncogenic signaling.

BACKGROUND: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR. METHODS: AR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively. RESULTS: The microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. CONCLUSION: Promotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.

A common 56-kilobase deletion in a primate-specific segmental duplication creates a novel butyrophilin-like protein.

BACKGROUND: The Butyrophilin-like (BTNL) proteins are likely to play an important role in inflammation and immune response. Like the B7 protein family, many human and murine BTNL members have been shown to control T lymphocytes response, and polymorphisms in human BTNL2 have been linked to several inflammatory diseases, such as pulmonary sarcoidosis, inflammatory bowel disease and neonatal lupus. RESULTS: In this study we provide a comprehensive population, genomic and transcriptomic analysis of a 56-kb deletion copy number variant (CNV), located within two segmental duplications of two genes belonging to the BTNL family, namely BTNL8 and BTNL3. We confirm the presence of a novel BTNL8*3 fusion-protein product, and show an influence of the deletion variant on the expression level of several genes involved in immune function, including BTNL9, another member of the same family. Moreover, by genotyping HapMap and human diversity panel (HGDP) samples, we demonstrate a clear difference in the stratification of the BTNL8_BTNL3-del allele frequency between major continental human populations. CONCLUSION: Despite tremendous progress in the field of structural variation, rather few CNVs have been functionally characterized so far. Here, we show clear functional consequences of a new deletion CNV (BTNL8_BTNL3-del) with potentially important implication in the human immune system and in inflammatory and proliferative disorders. In addition, the marked population differences found of BTNL8_BTNL3-del frequencies suggest that this deletion CNV might have evolved under positive selection due to environmental conditions in some populations, with potential phenotypic consequences.

Nanoarchaeal origin of histone H3?

NEQ288, one of two archaeal histones in Nanoarchaeum equitans, has a unique four-residue insertion that closely resembles an insertion in the eukaryotic histone H3 lineage. NEQ288 bound DNA but did not compact DNA in vitro in the absence of NEQ348, the second N. equitans archaeal histone. The properties of NEQ288 suggest an intermediate between the archaeal and H3 histone lineages and an evolutionary step toward the now-mandatory assembly of eukaryotic histones into heterodimers.

The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex.

Opitz BBB/G syndrome (OS) is a heterogenous malformation syndrome mainly characterised by hypertelorism and hypospadias. In addition, patients may present with several other defects of the ventral midline such as cleft lip and palate and congenital heart defects. The syndrome-causing gene encodes the X-linked E3 ubiquitin ligase MID1 that mediates ubiquitin-specific modification and degradation of the catalytic subunit of the translation regulator protein phosphatase 2A (PP2A). Here, we show that the MID1 protein also associates with elongation factor 1alpha (EF-1alpha) and several other proteins involved in mRNA transport and translation, including RACK1, Annexin A2, Nucleophosmin and proteins of the small ribosomal subunits. Mutant MID1 proteins as found in OS patients lose the ability to interact with EF-1alpha. The composition of the MID1 protein complex was determined by several independent methods: (1) yeast two-hybrid screening and (2) immunofluorescence, (3) a biochemical approach involving affinity purification of the complex, (4) co-fractionation in a microtubule assembly assay and (5) immunoprecipitation. Moreover, we show that the cytoskeleton-bound MID1/translation factor complex specifically associates with G- and U-rich RNAs and incorporates MID1 mRNA, thus forming a microtubule-associated ribonucleoprotein (RNP) complex. Our data suggest a novel function of the OS gene product in directing translational control to the cytoskeleton. The dysfunction of this mechanism would lead to malfunction of microtubule-associated protein translation and to the development of OS.

Active transport of the ubiquitin ligase MID1 along the microtubules is regulated by protein phosphatase 2A.

Mutations in the MID1 protein have been found in patients with Opitz BBB/G syndrome (OS), which is characterised by multiple malformations of the ventral midline. MID1 is a microtubule-associated protein that stabilizes microtubules and, in association with the regulatory subunit of protein phosphatase 2A (PP2A), alpha4, provides ubiquitin ligase activity for the ubiquitin-specific modification of PP2A. Using Fluorescence Recovery After Photobleaching (FRAP) technology, we show here that MID1 is actively and bi-directionally transported along the microtubules, and that this movement is directly linked to its MAP kinase and PP2A-mediated phosphorylation status. Intact transport depends on both kinesins and dyneins and is inhibited upon colcemide treatments. MID1 proteins carrying missense mutations in the alpha4 binding domain still bind the microtubules but cannot be actively transported. Likewise, knock-down of the alpha4 protein, inhibition of PP2A activity by okadaic acid and fostriecin or the simulation of permanent phosphorylation at Ser96 in MID1 stop the migration of MID1-GFP, while preserving its microtubule-association. In summary, our data uncover an unexpected and novel function for PP2A, its regulatory subunit alpha4 and PP2A/alpha4/mTOR signaling in the active transport of the MID1 ubiquitin ligase complex along the cytoskeleton. Furthermore, a failure in the microtubule directed transport of this protein complex would be an attractive mechanism underlying the pathogenesis of OS in patients with B-box1 mutations.